On the history of basic fuchsin and aldehyde-schiff reactions from 1862 to 1935

Summary The nature of products formed by aldehydes and Schiff's reagent, whether they are sulfonic or sulfinic acid compounds, has been the subject of much discussion. It seems therefore timely to review early studies of aldehyde-Schiff reactions, including the history of pararosanilin and related dyes. Dyes of the basic fuchsin group have been studied extensively since 1862, and their triphenylmethane structure was established in 1878. The currently used structural formulas were introduced around the turn of the century. Reactions of basic fuchsin with aldehydes, with and without addition of SO₂, were investigated by Schiff in the 1860's i.e. before the structure of these dyes was known. In 1900 Prud'homme showed that the reaction products of basic fuchsin, sodium bisulfite and formaldehyde are alkylated and sulfonated derivatives of the parent compound; further chemical studies indicated attachment of the sulfonic acid group to the carbon atom of the aldehyde. Prud'homme's findings were repeatedly confirmed during the following decades. Wieland and Scheuing were apparently unaware of these studies and introduced the sulfinic acid theory in 1921; furthermore, they considered substitution at two amino group of Schiff's reagent essential for formation of the colored compound. However, later chemical and spectroscopic studies showed no evidence of-N-sulfinic acids but supported the sulfonic acid theory of Prud'homme.     

Absence of Free Aldehydes in Fresh Tissues, as Shown with a Neutralized Schiff Reagent

A neutralized Schiff's reagent (pH 6.7) was prepared and used to investigate the role of the acidic nature of the routine Schiff's reagent (pH 2.6) in the plasmal reaction. The neutralized reagent was satisfactory as an aldehyde reagent in the nucleal reaction on gut and, although giving a less intense reaction than the routine reagent in the PAS reaction on the gut and plasmal reaction on the aorta, was satisfactory here in respect to localization and thus to aldehyde specificity. Control sections for the plasmal reaction of unfixed nerve and aorta gave positive results when placed in the routine Schiff's, this increasing with time left in the reagent. Similar control sections in the neutralized Schiff's reagent remained consistently negative even though left in this reagent for 0.5 hr. The positive reaction of such control sections is apparently due to acid hydrolysis of labile plasmalogens by the routine Schiff's reagent in myelin and elastin and not to the presence of “free” aldehydes in these tissue elements     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.     

On Schiff's bases and aldehyde-fuchsin: A review from H. Schiff to R.D. Lillie

Summary In histochemistry aldehyde-fuchsin is widely regarded as an azomethine compound, though this hypothesis cannot explain the variety of reaction products. Infrared spectroscopy did not show a C=N bond. It was therefore deemed of interest to review chemical studies of aldehydefuchsin and other Schiff's bases by Schiff and his contemporaries. Schiff regarded reaction products of low molecular aliphatic aldehydes, e.g. acetaldehyde, and aromatic amines as diarylamines; aldehyde-fuchsin was assigned a 23 (dyealdehyde) formula. These reactions were facilitated by alcohol and HCl. Others suggested condensation of two aldehyde molecules which carried a secondary and a tertiary amine respectively. Eibner proved that these compounds were ethylenes, not azomethines, and contained two secondary amines. Condensation of such bases produced ethylenic polymers, the Schultz's bases. Aromatic aldehydes readily yielded azomethines; aliphatic aldehydes formed –C=N– bonds only during prolonged heating. These findings are in agreement with recent chemical data. Clearly, the term Schiff's bases is not synonymous with azomethines, but denotes any reaction product of aldehydes and amines. In 1962, Lillie's histochemical studies confirmed the secondary amine nature of aldehyde aryl amine condensation. Thus, chemical and histochemical studies from Schiff in the 1860's to Lillie in the 1960's indicate that aldehyde-fuchsin is not an azomethine compound, but contains diarylamines and their derivatives.     

Studies in lipid histochemistry

Summary The control reaction with Schiff's reagent demonstrates besides free aldehydes also plasmalogens and is not a proper control test in lipid histochemistry in cases when reactions with Schiff's reagent are used in tissues containing plasmalogens. For the elimination of genuine aldehydes neutral blocking tests should be applied whenever plasmalogens are present.     

Histological and Histochemical Uses of Periodic Acid

Periodic acid acts upon the 1,2 glycol linkage (-CHOH -CHOH-) of carbohydrates in tissue sections to produce aldehyde (RCHO+RCHO) which can be colored with Schiff s reagent. The method can be used on frozen or paraffin sections and is useful as a reaction for carbohydrates of tissues: glycogen (in paraffin section only), mucin, basement membrane, reticulin, the colloid of the pituitary stalk and thyroid, some of the acidophile cells of the human anterior hypophysis, the granular cells of the renal arteriole, etc.

In abnormal tissues, it colors many of the “hyaline” materials— amyloid infiltrations, arteriolosclerotic hyaline, colloid droplets, mitotic figures, etc.

The histochemical uses of the periodic-acid-Schiff's reagent (PAS) need careful control because of the possibility of attachment of iodate or periodate to tissue constitutents, producing a recoloration of the Schiff's reagent. Whenever possible the positive reacting material should be further identified by other methods since Lison showed other substances besides aldehydes can recolorize SchifFs reagent.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

The chemical nature of de Tomasi Schiff reagent

Summary Samples of de Tomasi's Schiff reagent were allowed to age. During this process physical, chemical and staining properties of the reagent were monitored. Certain physical and chemical properties of Schiff's reagent correlated with staining intensities. Thus a fall in the iodine titre and a rise in the pH (indicating loss of sulphur dioxide) accompanied falls of intensity of Feulgen-stained frog red-cell nuclei and of P.A.S.-stained cellulose sheet. Ageing sulphite solutions exibited analogous physical and chemical changes. Thin layer and ion-exchange chromatography of Schiff's reagent demonstrated at least four colourless (but colour generating) derivatives, all of which were cationic. NMR spectroscopy showed that the carbon skeletons of these compounds were simple and symmetrical. The ageing of Schiff's reagent appears to be primarily due to changes in its inorganic components. The aldehyde-reactive compounds probably differ only in the nature of the oxysulphur substituent on their central carbon atom.     

Sequential staining of DNA-aldehyde with Schiff's reagent and acriflavine-SO2.

Acid hydrolysed DNA of rat liver was stained with Schiff's reagent at pHs 1.7 or 3.0 followed by staining with acriflavine-SO2 at pH 2.0 as well as with acriflavine-SO2 followed by Schiff's reagent at pH 1.7 or 3.0. Nuclei stained with Schiff's reagent at pH 1.7 were brown-yellow and an analysis of their absorption characteristics revealed two peaks--one at 470 nm and the other at 570 nm. Although nuclei stained with Schiff's reagent at pH 3.0 followed by acriflavine-SO2 were deep magenta in colour, they also showed similar peaks of maximum absorption. Identical peaks were also seen when the sequence of staining was reversed. It is suggested that in the conventional Feulgen-type reactions only some of the DNA-aldehyde molecules are stained; the remaining molecules can be stained by sequential application of another Schiff or Schiff-type reagent such as acriflavine-SO2. The possible mechanism of staining in these cases has been discussed.     

Effects of hydrogen ion dissociation and concentration of the reactivity of dichromate-fixed tissue components to the PAS procedure: recommendations for reducing undesirable background staining.

The undesirable PAS reactivity of cytoplasmic aldehydes after dichromate fixation can be suppressed without affecting selective staining by lowering the pH of Lillie's Cold Schiff reagent to 1.5. Alternatively, dilution of pH 2.2 Cold Schiff reagent with distilled water (1:2) is recommended. Hydrogen ion concentration and dissociation effect the rate of color formation in various PAS positive sites differentially with respect to the time of incubation in Schiff reagent. Based on the experiments, aldehydes exposed in different tissue components appear to be chemically distinct and separable depending on the rate of color formation and duration of incubation in Schiff reagent.     

Effects of Hydrogen Ion Dissociation and Concentration on The Reactivity of Dichromate-Fixed Tissue Components to the Pas Procedure: Recommendations for Reducing Undesirable Background Staining

The undesirable PAS reactivity of cytoplasmic aldehydes after dichromate fixation can be suppressed without affecting selective staining by lowering the pH of Lillie's Cold Schiff reagent to 1.5. Alternatively, dilution of pH 2.2 Cold Schiff reagent with distilled water (1:2) is recommended. Hydrogen ion concentration and dissociation affect the rate of color formation in various PAS positive sites differentially with respect to the time of incubation in Schiff reagent. Based on these experiments, aldehydes exposed in different tissue components appear to be chemically distinct and separable depending on the rate of color formation and duration of incubation in Schiff reagent.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

Studies on the Quantitative Nature of the Feulgen Stain

Bacterial suspensions were stained with Schiff's reagent according to the procedure suggested in essence by Dondero et al. (1954). Cell suspensions, Schiff's reagent, supernatant fluids and stained cells were analyzed by a micro-Kjeldahl procedure in an effort to quantitate the Feulgen reaction. The concentration of the bacterial suspension, type of fixative, time of hydrolysis and pH of cells and dye were varied and the effects analyzed quantitatively. While the cells were often stained deeply as determined by visual observation, the quantity of dye nitrogen in the cells was not large enough to be measured with the procedure employed. Significant quantitative results were obtained consistently only when the pH of the Schiff's reagent was raised. Feulgen reactions with solutions of formaldehyde and with solutions of DNA were also analyzed quantitatively after removing the colored compounds with charcoal. The analyses indicated that the DNA solution and the formaldehyde solution reacted differently with the dye.     

An Improved Lead-Tetraacetate:Schiff Procedure

Sections from tissues fixed in a 10% solution of formalin in 90% alcohol were treated with lead tetraacetate (PbAc₄) in different solvents: glacial acetic acid, dilute acetic acid, methanol, ethanol, toluene and benzene. The excess reagent was removed with a 2% solution of ethylene diamine tetraacetate (EDTA) at pH 8. The sections were then stained with Schiff's reagent for 20 minutes. The chemical stability of PbAc₄ in different solvents, the effect of its concentration and the time of exposure on the intensity of the Schiff reaction, were studied. A 0.023 N solution of PbAc₄ in benzene with a reaction time of 5 minutes is recommended. The stability of PbAc₄ in benzene permitted such solutions to be used for 3-4 days. Satisfactory results were obtained with mammalian tissues, algae, bacteria, fungi and protozoa. Some of the preparations obtained by using the technics described are illustrated in an accompanying plate of photomicrographs.     

Studies on the Preparation and Recoloration of Fuchsin Sulfurous Acid

Some of the factors affecting the recoloration of Schiff's Reagent (fuchsin sulfurous acid or FSA) by formaldehyde have been studied spectrophotometrically to determine the optimal conditions for the reaction of this reagent with aldehydes.

Of the various reducing agents utilized in the preparation of the leuco dye from basic fuchsin, sodium sulfite and bisulfite proved to be the most satisfactory for obtaining in the reagent maximal sensitivity to recoloration with minimal quantitative variation of results.

The relative proportions of reducing agent and basic fuchsin present in die leuco dye determine its sensitivity to recoloration. Under the conditions of the present experiments, greatest reagent recoloration was obtained when the leuco dye contained 0.01 mole of sodium bisulfite and 0.001 mole of basic fuchsin per 100 ml., a ratio of 10/1.

The recoloration of a given amount of FSA is related to the amount of aldehyde and the temperature of the reaction.

The present experiments indicate the desirability of standardizing the composition of FSA and the conditions under which it is used, if the results of different investigators are to be readily reproduced or compared.     

A new interpretation of the direct Schiff reaction of elastic connective tissue

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.     

A short-chain aldehyde is a major lipoxygenase product in arachidonic acid-stimulated porcine leukocytes

Porcine leukocytes convert exogenous arachidonic acid to a complex array of products derived via the 5-, 12-, and 15-lipoxygenase pathways of metabolism. The major monohydroxylated metabolite following addition of 100 microM arachidonic acid is 12-hydroxyeicosatetraenoic acid. Of the more polar compounds on reverse-phase high pressure liquid chromatography, the most prominent is a previously uncharacterized arachidonate product which chromatographs near to the omega-oxidized metabolites of leukotriene B4. The structure of this new product was examined by high pressure liquid chromatography, UV, NMR, and also by gas chromatography-mass spectrometry of several derivatives; it was identified as 12-oxododeca-5,8,10-(Z,Z,E)-trienoic acid. It is proposed that this C-12 trienal acid is formed from 12-hydroperoxyeicosatetraenoic acid by a cleavage reaction catalyzed by the leukocyte 12-lipoxygenase in the presence of excess arachidonic acid and under anaerobic conditions. These conditions are satisfied by addition of 100 microM arachidonic acid to the leukocyte suspension (3 X 10(7) cells/ml); 12-hydroperoxyeicosatetraenoic acid is formed as the major product, excess arachidonic acid is available, and the concomitant leukocyte respiratory burst quickly depletes the solution of oxygen. Preliminary experiments indicate that this aldehyde product has significant biological activity in the activation of leukocytes. In the course of an intense inflammatory reaction it is conceivable that the conditions for synthesis of this C-12 trienal acid and related aldehydes could prevail; such aldehydes would constitute an additional class of lipoxygenase product which exacerbates the process of inflammation.     

Autoradiographic thymidine-3H activity after successive steps of the Feulgen reaction.

Autoradiographs were prepared of sections of the ovary of Dytiscus marginalis labelled with thymidine-3H after each successive step of the Feulgen reaction and after treatment with each separate component of the Schiff's reagent. Results of grain counts over ovarian nurse cells showed that losses of thymidine-3H activity occur not only during hydrolysis but also during the successive steps of the Feulgen reaction. It is suggested that the latter decrease in radioactivity may depend on the extraction of fragments of apurinic acid from the sections. An emulsion desensitizing effect has also been observed in sections stained with basic fuchsin alone; this effect appears, however, to be strongly counteracted by the metabisulphite present in the Schiff's reagent.     

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.     

Comparative Spectroscopy of Basic Fuchsin, Schiff Reagent, and a Formalin-Schiff Derivative in Relation to Dye Structure and Staining

Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO₃H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl^- or SO₃H^- to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so reduced shows the fomation-NH₃ groups. Infrared (IR) spectra of basic fuchsin, Schiff crystals, and a crystalline formalin-Schiff reaction product support these observations and indicate that the final colored product is a methylsulfonic acid derivative of basic fuchsin. Identical IR spectra were obtained for two types of crystals derived from Schiff reagents indicating that both are the same chemically, although only one became colored on exposure to air. When these crystals were redissolved and SO₂ added, a Schiff reagent of appropriate pH was produced. Since it is derived from a crystalline product, this type of reagent should be useful in histochemical studies